We've been attempting to expand rat CD4+ CD25+ Foxp3+ regulatory T cells in vitro and have encountered difficulty - outgrowth of Foxp3- effector cells during expansion (>50% of cells) has been a major problem in most of our attempts.
Using rat (Lewis or Wistar Kyoto) splenocytes as the starting population, we have tried magnetic negative selection for CD4+ cells followed by FACS sorting for CD25hi cells. We have also tried magnetic positive selection for CD25+ cells followed by FACS sorting for CD4+ CD25hi cells. We start with a pure population that seems to correlate with Foxp3 expression on concomitant analytical flow cytometry. We expand the cells using plate-bound anti-CD3 (5ug/mL to coat) and soluble anti-CD28 (1ug/mL) with 300-1000 units per mL of recombinant human IL-2. We typically see a 30-60 fold expansion in 7 days, but most of the cells are usually Foxp3-. Addition of 1-100nM rapamycin to the culture medium has not helped.
I'm aware of the use of superagonistic CD28 antibodies by the Hunig group and others for this purpose, but was wondering whether anyone has had success using a more conventional anti-CD3/anti-CD28 approach?
Bona fide Treg surface markers are limited in the rat and I'm also wondering if part of the problem we are having is due to expression of Foxp3 in a CD4+ effector population that subsequently downregulates it upon expansion.
Your approach seems to be perfect for isolating Foxp3+ Treg cells. If you feel suitable, select CD4+CD25+CD127(LO) population as Treg population. Else below link may be helpful for you. All the best.
http://link.springer.com/protocol/10.1007%2F978-1-61737-979-6_2
Methods Mol Biol. 2011;707:21-37. doi: 10.1007/978-1-61737-979-6_2.
In vitro Treg suppression assays.
Collison LW1, Vignali DA.
Hi Mohammad, thanks for your answer. We tried CD127 but the only rat antibody available to us doesn't give much separation of positive and negative (or high and low) cells. What's more, among CD4+CD25hi cells, Foxp3+ expression was evenly distributed among CD127 high and low cells. Consequently, sorting on CD127 low cells has not really improved our expansion cultures. It may be that CD127 is not a as useful a marker in the rat as it is in the human.
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